The ultimate goal of this research is to characterize the interactions directing RNA folding, and to use this knowledge to predict reliably the structures of RNA molecules from their sequences. Since most diseases are mediated through RNA, and some viruses, including HIV, have RNA genomes with largely unknown secondary structures, our results can lead to rational design of therapeutics. The ability to predict RNA structure should also further the interpretation of sequences determined by the Human Genome Project and other sequencing efforts. Our results may also contribute to fighting bioterrorism because they potentially provide a rapid way to design therapeutics, including siRNA and antisense compounds, once a genome has been sequenced. The foundation for structure prediction will be advanced by studies of the thermodynamic and structural properties of oligonucleotides. Particular emphasis will be placed on the sequence dependence of stability and structure for internal loops. New collaborations with computational chemists will provide insight into the interactions determining both secondary and local three dimensional structure. Secondary structure prediction by free energy minimization will be augmented with constraints from sequence comparison, chemical mapping, oligonucleotide binding, and NMR to partially compensate for incomplete knowledge of the factors affecting folding. In particular, new microarray and nanoparticle methods will be developed to measure and interpret binding of chemically modified pentamers and hexamers to folded RNA. The foundation will be laid for design of a universal microarray that can be used to interrogate the structure of any RNA. The chemical mapping and microarray methods identify nucleotides that are not in Watson-Crick pairs. The new NMR assisted prediction of secondary structure (NAPSS) method uses unassigned spectra to further restrict folding space by identifying regions of canonical base pairs, including pseudoknots. The power of the predictive methods will be tested by attempting to determine the secondary structure of 323 nucleotides of the 5' coding region of an R2 retrotransposon. In 2006, it was discovered that this region of R2 RNA has a novel function of temporally orchestrating second strand DNA cleavage by R2 protein during insertion of the sequence into a genome. Initial results indicate that the RNA contains a novel pseudoknot. Project Narrative (Relevance) Most diseases are mediated through RNA, and some viruses, including HIV, have RNA genomes. The goal of this research is to be able to predict reliably the structure of an RNA from its sequence in order to provide a foundation for rational design of therapeutics targeting RNA. The results may also help fight bioterrorism because they potentially provide a rapid way to design therapeutics once a genome has been sequenced.